Despite growing evidence linking vitamin D to maternal and fetal health outcomes during pregnancy, little is known about the effect of pregnancy on vitamin D metabolism. Notably, the placenta is emerging as a possible source and regulator of circulating maternal vitamin D metabolites because it expresses all components of the vitamin D metabolic pathway and can produce 1,25[OH]2D which doubles during this reproductive state. Nonetheless, few studies have investigated the modulatory role of the placenta on maternal circulating vitamin D metabolites. In addition, although vitamin D is known to influence bone health in the nonpregnant state, it is unclear whether maternal vitamin D status influences maternal and fetal bone health during pregnancy. Animal studies have shown normal bone health outcomes in fetuses from severely vitamin D deficient mice while data from human studies are mixed possibly due to differences among studies in the dietary intakes of calcium, phosphorous, and other nutrients that impact bone health. This study seeks to advance understanding of vitamin D metabolism and requirements during pregnancy by measuring a comprehensive panel of vitamin D biomarkers from pregnant and nonpregnant control women who participated in a feeding study and consumed equivalent intakes of vitamin D and other related nutrients. The Specific Aims are as follows: Aim 1 will test the hypothesis that pregnancy alters blood biomarkers of vitamin D metabolism including 25(OH)D,1,25(OH)2D, 24,25(OH)2D and vitamin D binding protein (DBP). Aim 2 will test the hypothesis that placenta metabolizes vitamin D and contributes to the changes in maternal circulating vitamin D metabolites including 1,25(OH)2D. To achieve this aim, we will measure placental gene and protein expression levels of the megalin-cubilin receptor, 25-hydroxylase (CYP2R1), 1-hydroxylase (CYP27B1), and 24-hydroxylase (CYP24A1) as well as placental metabolite concentrations of 25(OH)D, 1,25(OH)2D, 24,25(OH)2D, and DBP. Relationships between placental and maternal biomarkers of vitamin D will be examined and a human placental cell culture model will be employed to investigate vitamin D metabolic flux and guide interpretation of the placental tissue experiments. Aim 3 will test the hypothesis that maternal serum 25(OH)D levels associate with maternal and fetal markers of bone metabolism. To achieve this aim, we will examine correlations between maternal markers of vitamin D status (i.e., 25[OH]D and 1,25[OH]2D) and maternal/cord blood levels of biochemical markers of bone health (e.g., parathyroid hormone).